Computational protocol: Stem Cell Transcription Factor FoxO Controls Microbiome Resilience in Hydra

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[…] For transcriptome sequencing transgenic lines were cocultured in shared HM with controls for at least four weeks in five independent replicates. After sampling, animals were frozen in TRIzol (Thermo Fisher Scientific) at -80°C until RNA extraction with the PureLink RNA Mini Kit (Ambion) according to the manufacturer’s protocol. Additionally, the optional on-column DNA digestion was performed. The RNA was eluted in 30 μl and checked for sufficient quality. If necessary, the RNA was purified using 1-butanol and diethyl ether () and frozen at -80°C until further use. Total RNA sequencing with previous ribosomal depletion was performed for 10 libraries on the Illumina HiSeq2500 v4 platform, with 125 bp paired-end sequencing of 12 libraries per lane. This resulted in 30–40 million reads per sample after quality control. Quality and adapters were trimmed using PRINSEQ-lite 0.20.4 (RRID:SCR_005454) () and Cutadapt 1.13 (RRID:SCR_011841) (). Subsequently, mapping against the H. vulgaris (AEP) transcriptome () was performed using Bowtie2 2.2.9 (RRID:SCR_005476) (). All downstream analyses were conducted in “R” (RRID:SCR_001905) (). Differentially expressed (DE) contigs were identified with the package DESeq2 1.16.1 (RRID:SCR_000154) (). The RNA-Seq raw data are deposited at the Sequence Read Archive (SRA) and are available under the project ID SRP133287. [...] Genomic DNA was extracted from individual polyps with the DNeasy Blood & Tissue Kit (Qiagen) as described in the manufacturer’s protocol. Elution was performed in 50 μl. Extracted DNA was stored at -20°C until sequencing. Prior to sequencing, the variable regions 1 and 2 (V1V2) of the bacterial 16S rRNA genes were amplified according to the previously established protocol using the primers 27F and 338R (). For bacterial 16S rRNA profiling, paired-end sequencing of 2 × 300 bp was performed on the Illumina MiSeq platform. The 16S rRNA sequencing raw data are deposited at the SRA and are available under the project ID SRP128106. The sequence analysis was conducted using the QIIME 1.9.0 package (RRID:SCR_008249) (). Paired-end reads were assembled using SeqPrep (RRID:SCR_013004). Chimeric sequences were identified with ChimeraSlayer (RRID:SCR_013283) () and verified manually before removal from the data set. If a putative chimera was present in at least two independent samples, the sequences were retained in the analysis. [...] Operational taxonomic unit (OTU) picking was performed using the protocol with at least 97% identity per OTU and annotation was conducted with the UCLUST algorithm (RRID:SCR_011921) () against the GreenGenes database v13.8 (RRID:SCR_002830) () implemented in QIIME. OTUs with <50 reads were removed from the data set to avoid false-positive OTUs that may originate from sequencing errors (). The number of reads was normalized to the lowest number of reads in the dataset. This was 22,000 reads for analyses including recolonizations with bacteria from H. vulgaris (AEP) and H. oligactis. For analyses including data from the H. viridissima source community the number of reads was normalized to 3,200 since reads assigned to chloroplasts of the algal endosymbiont were removed in silico. Alpha diversity was calculated using the Chao1 metric implemented in QIIME using ten replicates of rarefication per sample. Beta diversity was depicted in a PCoA by 100 jackknifed replicates using binary Pearson distances (BPDs). Bacterial groups specifically associated with control or FoxO- polyps were identified by LEfSe (RRID:SCR_014609) (). LEfSe couples robust tests for measuring statistical significance (Kruskal–Wallis test) with quantitative tests for biological consistency (Wilcoxon signed-rank sum test). The differentially abundant and biologically relevant bacterial groups are ranked by effect size after undergoing linear discriminant analysis. All p-values were corrected for multiple hypotheses testing using Benjamini and Hochberg’s false-discovery rate correction (q-value). A q-value of 0.25, an effect size threshold of 3.0 (on a log10 scale), and a mean abundance of at least 0.1% in one of the treatments were used for all bacterial groups discussed in this study. […]

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